US20110144393A1 - Production of butanediol by anaerobic microbial fermentation - Google Patents

Production of butanediol by anaerobic microbial fermentation Download PDF

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US20110144393A1
US20110144393A1 US12/997,063 US99706309A US2011144393A1 US 20110144393 A1 US20110144393 A1 US 20110144393A1 US 99706309 A US99706309 A US 99706309A US 2011144393 A1 US2011144393 A1 US 2011144393A1
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butanediol
substrate
mmol
fermentation
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Sean Dennis Simpson
Phuong Loan Tran
Christophe Daniel Mihalcea
Jennifer Mon Yee Fung
Fungmin Liew
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Lanzatech NZ Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to the production of butanediol by microbial fermentation, particularly to production of 2,3-butanediol by microbial fermentation of substrates comprising CO.
  • Biofuels for transportation are attractive replacements for gasoline and are rapidly penetrating fuel markets as low concentration blends.
  • Biofuels derived from natural plant sources, are more environmentally sustainable than those derived from fossil resources (such as gasoline), their use allowing a reduction in the levels of so-called fossil carbon dioxide (CO 2 ) gas that is released into the atmosphere as a result of fuel combustion.
  • CO 2 fossil carbon dioxide
  • biofuels can be produced locally in many geographies, and can act to reduce dependence on imported fossil energy resources.
  • Alcohols suitable for use as biofuels include ethanol, butanol and 2,3-butanediol.
  • Ethanol is rapidly becoming a major hydrogen-rich liquid transport fuel around the world.
  • Worldwide consumption of ethanol in 2002 was an estimated 10.8 billion gallons.
  • the global market for the fuel ethanol industry is also predicted to grow sharply in future, due to an increased interest in ethanol in Europe, Japan, the USA and several developing nations.
  • butanediols including 1,2-butanediol, 1,3-butanediol, 1,4-butanediol and 2,3-butanediol may be considered to have a variety of advantages over ethanol. Like ethanol, butanediols may be used directly as an automotive fuel additive. They may also be relatively easily transformed into a number of other potentially higher value and/or higher energy products. For example, 2,3-butanediol may be readily converted in a two step process into an eight-carbon dimer which can be used as aviation fuel.
  • 2,3-Butanediol derives its versatility from its di-functional backbone, i.e., 2 hydroxyl groups are located at vicinal C-atoms allowing the molecule to be transformed quite easily into substances such as butadiene, butadione, acetoin, methylethyl ketone etc. These chemical compounds are used as base molecules to manufacture a vast range of industrially produced chemicals.
  • 2,3-butanediol may be used as a fuel in an internal combustion engine. It is in several ways more similar to gasoline than it is to ethanol. As the interest in the production and application of environmentally sustainable fuels has strengthened, interest in biological processes to produce 2,3-butanediol (often referred to as bio-butanol) has increased.
  • 2,3-Butanediol can be produced by microbial fermentation of carbohydrate containing feedstock (Syu M J, Appl Microbial Biotechnol 55:10-18 (2001), Qin et al., Chinese J Chem Eng 14(1):132-136 (2006)).
  • 2,3-Butanediol may also be produced by microbial fermentation of biomass from crops such as sugar beet, corn, wheat and sugarcane.
  • crops such as sugar beet, corn, wheat and sugarcane.
  • the cost of these carbohydrate feed stocks is influenced by their value as human food or animal feed and the cultivation of starch or sucrose-producing crops for 2,3-butanediol production is not economically sustainable in all geographies. Therefore, it is of interest to develop technologies to convert lower cost and/or more abundant carbon resources into 2,3-butanediol.
  • Carbon Monoxide is a major by-product of the incomplete combustion of organic materials such as coal or oil and oil derived products. Although the complete combustion of carbon containing precursors yields CO2 and water as the only end products, some industrial processes need elevated temperatures favouring the build up of carbon monoxide over CO2.
  • One example is the steel industry, where high temperatures are needed to generate desired steel qualities. For example, the steel industry in Australia is reported to produce and release into the atmosphere over 500,000 tonnes of CO annually.
  • syngas is also a major component of syngas, where varying amounts of CO and H2 are generated by gasification of a carbon-containing fuel.
  • syngas may be produced by cracking the organic biomass of waste woods and timber to generate precursors for the production of fuels and more complex chemicals.
  • CO is a reactive energy rich molecule, it can be used as a precursor compound for the production of a variety of chemicals. However, this valuable feedstock has not been utilised to produce 2,3-butanediol.
  • the invention provides a method of producing butanediol by microbial fermentation of a substrate comprising carbon monoxide.
  • the invention provides a method of producing butanediol by microbial fermentation, the method including:
  • the butanediol is 2,3-butanediol.
  • the invention provides a method of increasing efficiency of 2,3-butanediol production by fermentation, the method including:
  • a method of producing 2,3-butanediol by microbial fermentation including:
  • the substrate comprises CO.
  • the substrate comprising carbon monoxide is a gaseous substrate comprising carbon monoxide.
  • the gaseous substrate comprising carbon monoxide can be obtained as a by-product of an industrial process.
  • the industrial process is selected from the group consisting of ferrous metal products manufacturing, non-ferrous products manufacturing, petroleum refining processes, gasification of biomass, gasification of coal, electric power production, carbon black production, ammonia production, methanol production and coke manufacturing.
  • the gaseous substrate comprises a gas obtained from a steel mill.
  • the gaseous substrate comprises automobile exhaust fumes.
  • the CO-containing substrate typically contains a major proportion of CO, such as at least about 20% to about 100% CO by volume, from 40% to 95% CO by volume, from 40% to 60% CO by volume, and from 45% to 55% CO by volume.
  • the substrate comprises about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% CO, or about 55% CO, or about 60% CO by volume.
  • Substrates having lower concentrations of CO, such as 6%, may also be appropriate, particularly when H 2 and CO 2 are also present.
  • the substrate comprising CO is provided at a sufficient level, such that 2,3-butanediol is produced.
  • CO is provided such that a specific uptake rate of at least 0.4 mmol/g/min; or at least 0.5 mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7 mmol/g/min; or at least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; or at least 1.0 mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5 mmol/g/min is maintained.
  • the method comprises microbial fermentation using Clostridium autoethanogenum.
  • the invention provides a method of producing 2,3-butanediol by microbial fermentation, the method including:
  • the substrate is one or more carbohydrates such as fructose.
  • the substrate is a substrate comprising carbon monoxide, typically a gaseous substrate comprising carbon monoxide, as herein before described
  • the invention provides a method for producing butanediol by microbial fermentation of a first substrate and a second substrate comprising CO.
  • the butanediol is 2,3-butanediol.
  • the first substrate is a carbohydrate.
  • the first substrate is fructose.
  • the second substrate is a gaseous substrate comprising carbon monoxide, as herein before described.
  • the method includes the steps of:
  • steps (a) and (b) may be conducted at the same time.
  • step (a) may substantially precede or follow step (b).
  • the method may alternate between step (a) and step (b).
  • butanediol preferably 2,3-butanediol, produced by the methods of any of the previous aspects.
  • the invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
  • butanediol refers to all structural isomers of the diol including 1,2-butanediol, 1,3-butanediol, 1,4-butanediol and 2,3-butanediol and stereoisomers thereof.
  • 2,3-butanediol should be interpreted to include all enantiomeric and diastereomeric forms of the compound, including (R,R), (S,S) and meso forms, in racemic, partially stereoisomerically pure and/or substantially stereoisomerically pure forms.
  • bioreactor includes a fermentation device consisting of one or more vessels and/or towers or piping arrangement, which includes the Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, Static Mixer, or other vessel or other device suitable for gas-liquid contact.
  • CSTR Continuous Stirred Tank Reactor
  • ICR Immobilized Cell Reactor
  • TBR Trickle Bed Reactor
  • Bubble Column Gas Lift Fermenter
  • Static Mixer Static Mixer
  • substrate comprising carbon monoxide and like terms should be understood to include any substrate in which carbon monoxide is available to one or more strains of bacteria for growth and/or fermentation, for example.
  • Gaseous substrates comprising carbon monoxide include any gas which contains a level of carbon monoxide.
  • the gaseous substrate will typically contain a major proportion of CO, preferably at least about 15% to about 95% CO by volume.
  • 2,3-butanediol can be produced by microbial fermentation using Clostridium autoethanogenum . They have found that fermentation products include a variety of alcohols, whereby ethanol and 2,3-butanediol are significant substituents. 2,3-Butanediol has not been previously identified as a fermentation product using Clostridium autoethanogenum . In particular, the inventors have determined that Clostridium autoethanogenum can be used to produce 2,3-butanediol and other products from a substrate comprising carbohydrate. In particular, fructose can be converted into products including acetate, ethanol and 2,3-butanediol.
  • 2,3-butanediol can be produced by Clostridium autoethanogenum from substrates comprising CO, particularly gaseous substrates comprising CO.
  • a gaseous carbon source particularly a source including CO
  • fermentation processes has not previously resulted in the production of 2,3-butanediol.
  • the efficiency of 2,3-butanediol production can be increased by providing the substrate at a sufficient level such that 2,3-butanediol is produced. It has been recognised that increasing the amount of substrate provided to a microbial culture, increases the amount of 2,3-butanediol produced by the culture.
  • the substrate comprising CO is provided at a sufficient level such that 2,3-butanediol is produced. It has been shown that a microbial culture comprising C. autoethanogenum can uptake CO at a rate up to approximately 1.5 to 2 mmol/gram dry weight microbial cells/minute (specific CO uptake). In particular embodiments of the invention, a substrate comprising CO is provided to the microbial culture comprising C.
  • 2,3-butanediol is a significant fermentation product of at least 0.5 g/L; or at least 1 g/L; or at least 2 g/L; or at least 5 g/L. In particular embodiments, 2,3-butanediol is produced at a rate of at least 0.5 g/L/day; or at least 1 g/L/day.
  • apparatus used for conducting methods of the invention enable measurement and/or control of parameters such as CO supply, CO uptake, biomass level, 2,3-butanediol production.
  • parameters such as CO supply, CO uptake, biomass level, 2,3-butanediol production.
  • samples can be taken from a bioreactor to determine one or more of the above parameters and the bioreactor conditions optionally adjusted to improve 2,3-butanediol production.
  • the CO supply can be increased such that 2,3-butanediol is produced.
  • 2,3-butanediol can be produced, particularly where CO is provided such that specific CO uptake rates of at least 0.4 mmol/g/min; or at least 0.5 mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7 mmol/g/min; or at least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; or at least 1.0 mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5 mmol/g/min are maintained.
  • C. autoethanogenum a-acetolactate synthase (ALS), ⁇ -acetolactate decarboxylase (ALDC) and 2,3-butanediol dehydrogenase (2,3BDH).
  • ALS a-acetolactate synthase
  • ADC ⁇ -acetolactate decarboxylase
  • 2,3BDH 2,3-butanediol dehydrogenase gene (ORF 1283) of C. autoethanogenum (strain deposited at DSMZ under the accession number 19630) shows strong homology to the 2,3BDH of Clostridium novyi (NT01CX — 0344) with amino acid identities of 73% (262/357) and positives of 84% (300/357).
  • 2,3-butanediol is produced from pyruvate (an intermediate in anabolism produced from acetyl CoA) as follows:
  • 2,3-butanediol dehydrogenase can be upregulated in accordance with the methods of the invention. For example, where CO is supplied at sufficient levels, 2,3-butanediol dehydrogenase is upregulated.
  • the specific CO uptake by the microbial culture is at least 0.4 mmol/g/min; or at least 0.5 mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7 mmol/g/min; or at least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; or at least 1.0 mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5 mmol/g/min; 2,3-butanediol dehydrogenase is upregulated.
  • the invention provides a method of producing 2,3-butanediol by microbial fermentation of a substrate by upregulation of 2,3-butanediol dehydrogenase.
  • substrates such as a carbohydrate substrate and a gaseous substrate comprising CO
  • substrates can be switched during microbial production of 2,3-butanediol, without deleterious effect.
  • substrates could be alternated, for example when one substrate is unavailable, and would continue to produce 2,3-butanediol.
  • 2,3-butanediol is produced by microbial fermentation of a substrate comprising carbohydrate.
  • a substrate comprising carbon monoxide preferably a gaseous substrate comprising CO, is converted into various products including 2,3-butanediol, by Clostridium autoethanogenum.
  • a first substrate comprising carbohydrate (preferably fructose) may be used in initial stages of the fermentation reaction and following complete consumption of the substrate, the substrate can be switched to a second substrate comprising CO.
  • carbohydrate preferably fructose
  • the inventors have surprisingly determined that 2,3-butanediol is produced in the initial stages where the first substrate comprising carbohydrate is the sole carbon source and is also produced in the latter stages where the substrate comprising CO is the sole carbon source.
  • 2,3-butanediol is produced under a variety of conditions, including media containing alternative buffer solutions such as acetate buffer and citrate buffer.
  • media containing alternative buffer solutions such as acetate buffer and citrate buffer.
  • the inventors also submit that in embodiments where the pH is uncontrolled and may be variable, 2,3-butanediol is still produced. Examples of media suitable for carrying out the desired fermentation are described in the examples section hereinafter.
  • the inventors contemplate that the 2,3-butanediol produced in such processes may be readily recovered using separation techniques known in the art. Furthermore, the 2,3-butanediol may be readily converted into substances such as butadiene, butadione, acetoin, methylethyl ketone and the like. Such chemical compounds are valuable base molecules used to manufacture a significant percentage of all chemical industry products. Therefore, the inventors contemplate that the 2,3-butanediol produced in the processes disclosed herein may be used in the manufacture of a wide range of well known industrial products.
  • the invention provides a method for the production of butanediol by microbial fermentation.
  • the method comprises at least the step of anaerobically fermenting a substrate comprising CO, preferably a gaseous substrate comprising CO, to obtain 2,3-butanediol.
  • the method includes the steps of:
  • the invention provides a method of increasing efficiency of 2,3-butanediol production by fermentation, the method including:
  • the substrate comprising CO is provided at a level sufficient to produce significant amounts of 2,3-butanediol, such as at least 0.5 g/L of fermentation media, or at least 1 g/L, or at least 2 g/L, or at least 5 g/L.
  • CO is provided at a level sufficient to produce 2,3-butanediol at a rate of at least 0.5 g/L/day; or at least 1 g/L/day.
  • CO is provided such that a specific uptake rate of at least 0.4 mmol/g/min; or at least 0.5 mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7 mmol/g/min; or at least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; or at least 1.0 mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5 mmol/g/min is maintained.
  • a specific uptake rate of at least 0.4 mmol/g/min; or at least 0.5 mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7 mmol/g/min; or at least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; or at least 1.0 mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5 mmol/g/min is maintained
  • gas hold-up in a fermentation media will increase the amount of CO available for conversion to products by the microbial culture.
  • Gas hold-up can typically be increased by mechanical means, such as increasing agitation in a CSTR.
  • supplying CO at a faster rate or a higher partial pressure will also increase the CO availability in a fermentation broth.
  • the method involves fermentation of a substrate comprising carbohydrate by Clostridium autoethanogenum to produce butanediol, preferably, 2,3-butanediol.
  • the method includes the steps of:
  • the first substrate is carbohydrate and in some embodiments, the substrate is fructose.
  • the second substrate is a gaseous substrate comprising CO.
  • steps (a) and (b) may be conducted at the same time.
  • step (a) may substantially precede or follow step (b).
  • the method may alternate between step (a) and step (b).
  • the method further includes the step of capturing or recovering the 2,3-butanediol produced.
  • the one or more micro-organisms used in the fermentation is Clostridium autoethanogenum .
  • the Clostridium autoethanogenum is a Clostridium autoethanogenum having the identifying characteristics of the strain deposited at the German Resource Centre for Biological Material (DSMZ) under the identifying deposit number 19630.
  • the Clostridium autoethanogenum is a Clostridium autoethanogenum having the identifying characteristics of DSMZ deposit number DSMZ 10061.
  • Culturing of the bacteria used in a method of the invention may be conducted using any number of processes known in the art for culturing and fermenting substrates using anaerobic bacteria. Exemplary techniques are provided in the “Examples” section of this document. By way of further example, those processes generally described in the following articles using gaseous substrates for fermentation may be utilised: K. T. Klasson, M. D. Ackerson, E. C. Clausen and J. L. Gaddy (1991). Bioreactors for synthesis gas fermentations resources. Conservation and Recycling, 5; 145-165; K. T. Klasson, M. D. Ackerson, E. C. Clausen and J. L. Gaddy (1991). Bioreactor design for synthesis gas fermentations. Fuel.
  • 2,3-butanediol is produced by microbial fermentation of a substrate comprising carbohydrate using Clostridium autoethanogenum .
  • suitable substrates may include, but are not limited to, monosaccharides such as glucose and fructose, oligosaccharides such as sucrose or lactose, polysaccharides, such as cellulose or starch.
  • monosaccharides such as glucose and fructose
  • oligosaccharides such as sucrose or lactose
  • polysaccharides such as cellulose or starch.
  • preferred carbohydrate substrates are fructose and sucrose (and mixtures thereof).
  • fermentable sugars may be obtained from cellulosic and lignocellulosic biomass through processes of pre-treatment and saccharification, as described, for example, in US20070031918.
  • Biomass refers to any cellulose or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides.
  • Biomass includes, but is not limited to bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. However, in exemplary embodiments of the invention commercially available fructose is used as the carbon and energy source for the fermentation.
  • a substrate comprising carbon monoxide preferably a gaseous substrate comprising carbon monoxide is used in the methods of the invention.
  • the gaseous substrate may be a waste gas obtained as a by-product of an industrial process, or from some other source such as from combustion engine (for example automobile) exhaust fumes.
  • the industrial process is selected from the group consisting of ferrous metal products manufacturing, such as a steel mill, non-ferrous products manufacturing, petroleum refining processes, gasification of coal, electric power production, carbon black production, ammonia production, methanol production and coke manufacturing.
  • the CO-containing gas may be captured from the industrial process before it is emitted into the atmosphere, using any convenient method.
  • the gaseous substrate may also be desirable to treat it to remove any undesired impurities, such as dust particles before introducing it to the fermentation.
  • the gaseous substrate may be filtered or scrubbed using known methods.
  • the gaseous substrate comprising carbon monoxide may be sourced from the gasification of biomass.
  • the process of gasification involves partial combustion of biomass in a restricted supply of air or oxygen.
  • the resultant gas typically comprises mainly CO and H 2 , with minimal volumes of CO 2 , methane, ethylene and ethane.
  • biomass by-products obtained during the extraction and processing of foodstuffs such as sugar from sugarcane, or starch from maize or grains, or non-food biomass waste generated by the forestry industry may be gasified to produce a CO-containing gas suitable for use in the present invention.
  • the CO-containing substrate will typically contain a major proportion of CO, such as at least about 20% to about 100% CO by volume, from 40% to 95% CO by volume, from 40% to 60% CO by volume, and from 45% to 55% CO by volume.
  • the substrate comprises about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% CO, or about 55% CO, or about 60% CO by volume.
  • Substrates having lower concentrations of CO, such as 6%, may also be appropriate, particularly when H 2 and CO 2 are also present.
  • CO is supplied at a level sufficient for 2,3-butanediol production to occur.
  • CO is provided such that a specific uptake rate of at least 0.4 mmol/g/min; or at least 0.5 mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7 mmol/g/min; or at least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; or at least 1.0 mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5 mmol/g/min is maintained.
  • Those skilled in the art will appreciate methods of supplying CO, particularly gaseous CO, such that the required uptake rate is achieved.
  • gas hold-up in a fermentation media will increase the amount of CO available for conversion to products by the microbial culture.
  • gas hold-up is typically increased by mechanical means such as increasing agitation in a CSTR.
  • supplying CO at a faster rate or a higher partial pressure will also increase the CO availability in a fermentation broth.
  • the gaseous substrate may also contain some CO 2 for example, such as about 1% to about 80% by volume, or 1% to about 30% by volume. In one embodiment it contains about 5% to about 10% by volume. In another embodiment the gaseous substrate contains approximately 20% CO 2 by volume.
  • the carbon monoxide will be added to the fermentation reaction in a gaseous state.
  • the invention should not be considered to be limited to addition of the substrate in this state.
  • the carbon monoxide could be provided in a liquid.
  • a liquid may be saturated with a carbon monoxide containing gas and then that liquid added to a bioreactor. This may be achieved using standard methodology.
  • a microbubble dispersion generator Hensirisak et. al. Scale-up of microbubble dispersion generator for aerobic fermentation; Applied Biochemistry and Biotechnology Volume 101, Number 3/October, 2002
  • a microbubble dispersion generator Heensirisak et. al. Scale-up of microbubble dispersion generator for aerobic fermentation; Applied Biochemistry and Biotechnology Volume 101, Number 3/October, 2002
  • 2,3-butanediol can be produced by fermentation of a first substrate and a second substrate.
  • 2,3-butanediol will be produced when a first substrate, for example a carbohydrate such as fructose and a second substrate, preferably a substrate comprising CO, are provided.
  • the inventors have determined that 2,3-butanediol will be produced by a first substrate and on complete consumption, the first substrate may be replaced with a second substrate and the 2,3-butanediol continues to be produced.
  • the first substrate is fructose and on complete consumption of the fructose, a substrate comprising CO can be provided.
  • the inventors have surprisingly found that 2,3-butanediol continues to be produced.
  • the first substrate and second substrate may be alternated if needed. For example if a first substrate is unavailable, an alternative substrate may be used until the availability of the first substrate improves.
  • a suitable nutrient medium will need to be fed to the bioreactor.
  • a nutrient medium will contain components, such as vitamins and minerals, sufficient to permit growth of the micro-organism used.
  • Anaerobic media suitable for the growth of Clostridium autoethanogenum are known in the art, as described for example by Abrini et al (Clostridium autoethanogenum, sp. Nov., An Anaerobic Bacterium That Produces Ethanol From Carbon Monoxide; Arch. Microbiol ., 161: 345-351 (1994)).
  • the “Examples” section herein after provides further examples of suitable media.
  • the fermentation should desirably be carried out under appropriate conditions for the substrate to butanediol fermentation to occur.
  • Reaction conditions that should be considered include temperature, media flow rate, pH, media redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum substrate concentrations and rates of introduction of the substrate to the bioreactor to ensure that substrate level does not become limiting, and maximum product concentrations to avoid product inhibition.
  • Examples of fermentation conditions suitable for anaerobic fermentation of a substrate comprising CO are detailed in WO2007/117157, WO2008/115080, WO2009/022925 and WO2009/064200, the disclosure of which are incorporated herein by reference. It is recognised the fermentation conditions reported therein can be readily modified in accordance with the methods of the instant invention.
  • the inventors have determined that, in one embodiment where pH is not controlled, there does not appear to be a deleterious effect on 2,3-butanediol production.
  • the bioreactor may comprise a first, growth reactor in which the micro-organisms are cultured, and a second, fermentation reactor, to which broth from the growth reactor is fed and in which most of the fermentation product (2,3-butanediol, for example) is produced.
  • the fermentation will result in a fermentation broth comprising a desirable product (such as butanediol) and/or one or more by-products (such as ethanol, acetate and butyrate) as well as bacterial cells, in a nutrient medium.
  • a desirable product such as butanediol
  • one or more by-products such as ethanol, acetate and butyrate
  • bacterial cells in a nutrient medium.
  • the fermentation products include 2,3-butanediol.
  • 2,3-butanediol, or a mixed alcohol stream containing 2,3-butanediol and one or more other alcohols may be recovered from the fermentation broth by methods known in the art, such as fractional distillation or evaporation, pervaporation, and extractive fermentation.
  • By-products such as acids including acetate and butyrate may also be recovered from the fermentation broth using methods known in the art.
  • an adsorption system involving an activated charcoal filter or electrodialysis may be used.
  • 2,3-butanediol and by-products are recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating microbial cells from the broth (conveniently by filtration, for example), and recovering 2,3-butanediol and optionally other alcohols and acids from the broth.
  • Alcohols may conveniently be recovered for example by distillation, and acids may be recovered for example by adsorption on activated charcoal.
  • the separated microbial cells are preferably returned to the fermentation bioreactor.
  • the cell free permeate remaining after the alcohol(s) and acid(s) have been removed is also preferably returned to the fermentation bioreactor. Additional nutrients (such as B vitamins) may be added to the cell free permeate to replenish the nutrient medium before it is returned to the bioreactor.
  • the pH of the broth was adjusted during recovery of 2,3-butanediol and/or by-products, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor.
  • Solution A NH 4 Ac 3.083 g KCl 0.15 g MgCl 2 •6H 2 O 0.61 g NaCl (optional) 0.12 g CaCl 2 •2H 2 O 0.294 g Distilled Water Up to 1 L Solution B Biotin 20.0 mg Calcium D-(*)- 50.0 mg pantothenate Folic acid 20.0 mg Vitamin B12 50.0 mg Pyridoxine. HCl 10.0 mg p-Aminobenzoic acid 50.0 mg Thiamine.
  • a 1 L three necked flask was fitted with a gas tight inlet and outlet to allow working under inert gas and subsequent transfer of the desired product into a suitable storage flask.
  • the flask was charged with CrCl 3 .6H 2 O (40 g, 0.15 mol), zinc granules [20 mesh] (18.3 g, 0.28 mol), mercury (13.55 g, 1 mL, 0.0676 mol) and 500 mL of distilled water. Following flushing with N 2 for one hour, the mixture was warmed to about 80° C. to initiate the reaction. Following two hours of stirring under a constant N 2 flow, the mixture was cooled to room temperature and continuously stirred for another 48 hours by which time the reaction mixture had turned to a deep blue solution. The solution was transferred into N 2 purged serum bottles and stored in the fridge for future use.
  • Clostridium autoethanogenum used is that deposited at the German Resource Centre for Biological Material (DSMZ) and allocated the accession number 19630.
  • Media samples were taken from the fermentation reactor (e.g. CSTR or serum bottle) at intervals over the course of the fermentation. Each time the media was sampled care was taken to ensure that no gas was allowed to enter into or escape from the reactor.
  • the fermentation reactor e.g. CSTR or serum bottle
  • Channel 1 was a 10 m Mol-sieve column running at 70° C., 200 kPa argon and a backflush time of 4.2 s
  • channel 2 was a 10 m PPQ column running at 90° C., 150 kPa helium and no backflush.
  • the injector temperature for both channels was 70° C.
  • Runtimes were set to 120 s, but all peaks of interest would usually elute before 100 s.
  • Specific CO uptake was determined by calculating CO consumption per gram of cells (dry weight—see below).
  • Cell density was determined by counting bacterial cells in a defined aliquot of fermentation broth. Alternatively, the absorbance of the samples was measured at 600 nm (spectrophotometer) and the dry weight determined via calculation according to published procedures.
  • the media used for the CSTR experiments were prepared in accordance with the components listed in Table E.
  • the phosphate salt mixture consisted of 0.65 mM Na 2 HPO4 and 15.3 mM NaH 2 PO 4 . All other components such as the phosphoric acid, the ammonium salts and the cysteine-hydrochloride were mixed into 800 ml of water before the buffer salts were added to the solution. Proceeding in this manner ensured that the pH increased above about 6.5 avoiding the precipitation of media components.
  • the solution was diluted to 1 L and made anaerobic by heating to boiling and allowing it to cool to room temperature under a constant flow of N2 gas. Once cool, the solution was adjusted to the final pH of 5.3 and the B vitamins added. Anaerobicity was maintained throughout the experiment. Carbohydrate (5 g/L fructose) was added to the basic media formulation. The media solutions were introduced into the fermenters and optionally sparged with the respective CO containing gases from the start of the experiment, or after a predetermined interval. During these experiments, the pH was controlled to remain at 5.5 by adding an aqueous solution of NaOH. An actively growing Clostridium autoethanogenum culture was inoculated into the reactor at a level of 5% (v/v). The temperature of the reactor was maintained at 37° C. and agitation rate was 400 rpm.
  • the fermentation contained fructose as a substrate, which resulted in the production of acetic acid, ethanol and 2,3-butanediol.
  • fructose was consumed and a gas stream including CO (95% CO, 5% CO2) was sparged through the media.
  • the media was maintained at pH 5.5 (Table 1). It should be noted that even when the carbohydrate had been consumed, the above mentioned products increased in concentration, clearly demonstrating that the CO was used to produce the products including 2,3-butanediol.
  • Total 2,3-butanediol accumulation over 7.5 days was approx 7.5 g/L. It is recognised that 2,3-butanediol is produced at low levels at lower specific CO uptake rates. However, when the gas is supplied such that the CO uptake rate can be maintained over 0.4 mmol/g/min, 2,3-butanediol productivity increase significantly. In this instance, the specific CO uptake is maintained at an average of 0.8 mmol/g/min over several days and 1,3-butanediol is produced at a rate in excess of 1 g/L.
  • Total 2,3-butanediol concentration after 4 days was approximately 3 g/L. While the rates achieved are less than previous fermentations (examples 4 and 5), the substrate stream comprises a substantial portion of hydrogen. The results show that 2,3-butanediol is produced when using a mixed CO/H2 substrate.
  • a five-litre bioreactor was charged with 4.9 L of LM33 media prepared as described above.
  • the gas was switched to CO containing gas (1% H2; 14% N2; 70% CO; 15% CO2) at atmospheric pressure prior to inoculation with 100 ml of a Clostridium autoethanogenum culture.
  • the bioreactor was maintained at 37° C. stirred at 200 rpm at the start of the culture. During the growth phase, the agitation was increased to 400 rpm.
  • the pH was adjusted to 5.5 and maintained by automatic addition of 5 M NaOH. Fresh anaerobic media was continuously added into the bioreactor to maintain a defined biomass and acetate level in the bioreactor. 2,3 butanediol productivity is highlighted in Table 9.
  • the fermenter was operated under CO limited conditions and minimal 2,3-butanediol was produced.
  • gas flow was increased, such that specific CO uptake increased.
  • 2,3-butanediol productivity increased significantly to at least 1.2 g/L/day.
  • the gas flow was reduced such that the specific uptake of the culture decreased to around 0.4 mmol/g/min and the 2,3-butanediol productivity also dropped.
  • 2,3-butanediol productivity remained at least 0.5 g/L/day.
  • Samples were taken from three fermentations to determine gene expression during 2,3-butanediol production.
  • One sample was taken from the batch fermentation described in Example 8 on day 13, wherein products including ethanol and 2,3-butanediol were being produced.
  • the sample is designated R12 in the results hereinafter.
  • the second sample was taken from a batch fermentation producing both ethanol and 2,3-butanediol.
  • the sample is designated R11 in the results.
  • the third sample (R2) was taken from the continuous fermentation operating under similar conditions as Example 7 on days 1-89.
  • the microbial culture was CO limited and the fermentation broth had a stable acetate concentration of approximately 13 g/L, ethanol concentration of less than 1 g/L and insignificant amounts of 2,3-butanediol.
  • Real-Time PCR was used to determine whether genes were upregulated or downregulated relative to R2.
  • Primers for Real-Time PCR were designed using the freeware Primer3 based on LanzaTech's proprietary in-house genome sequence.
  • Real-Time PCR reaction mixtures containing 12.5 ⁇ L 2 ⁇ SYBR Green PCR Master Mix (Biorad), 1.54 of each of 1 ⁇ M primer forward and reverse, 5 ⁇ L of 10 ⁇ diluted cDNA template, and sterile water to a total volume of 25 ⁇ L were assembled. The mixtures were heated to 95° C. for 3 minutes, followed by 40 cycles of 95° C. for 15 seconds, 55° C. for 15 seconds and 72° C. for 30 seconds.
  • RNA polymerase beta chain (rpoB) was selected as reference gene for normalizing gene expression. Relative quantification using the Comparative ⁇ C T method was used to calculate the relative gene expression of 2,3BDH.
  • the acetate-producing culture (R2) was selected as calibrator (reference standard) in all analysis.
  • FIG. 1 shows relative gene expression of 2,3-butanediol dehydrogenase (2,3BDH) in three fermenters (R11, R12 and R2).
  • 2,3-butanediol dehydrogenase is upregulated in microbial cultures that produce 2,3-butanediol.
  • the microbial culture in R2 has a specific CO uptake of approximately 0.3 mmol/g/min, whereas the culture in R12 has a specific uptake of approximately 0.6 mmol/g/min.

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